This R33 project addresses the ongoing demand for new imaging tools capable of resolving the nanoscale organization and architecture of signal transduction machinery in living cells. Physical forces generated by and acting on cells and tissues influence how tumors develop and metastasize. These forces are mediated through receptor assemblies on the cell membrane that convert the mechanical stimulus into biochemical signals, which directly affect cellular growth, survival, and migration. In this projec, a new hardware platform will be constructed and validated that significantly expands the capabilities of Scanning Angle Interference Microscopy (SAIM), an optical imaging methodology previously developed by the researchers. SAIM combines interferometry and fluorescence microscopy into an imaging modality capable of imaging fluorescently labeled structures in living cells with 10-nm axial resolution. The innovative re-design of SAIM proposed in this project will address its biggest limitations: slow imaging speeds, poor signal-to-background in thick specimens, and limited versatility in regards to the cell culture platform. The instrument developed will enable sub-second nanoscale imaging of living cells on or embedded within thin hydrogels. In addition, the imaging depth of SAIM will be further extended by implementing localized fluorophore activation that restricts the fluorescence to a thin plane using two-photon photoactivation and uncaging, therefore avoiding background from labeled cells outside of our region of interest. Instrument validation studies will be directed toward understanding how integrin-based signaling complexes sense and respond to cancer-relevant mechanical forces. The proposed combination of axial localization precision and dynamic, live-cell capability will be unique among super- resolution technologies. Such an instrument would find uses in many other areas of biomedical research ranging from studies of viral fusion and assembly on membranes to high-resolution studies of the 3D structure of chromatin within the nucleus. By the end of the R33 funding period, a new and robust imaging modality will have been created that has been fully validated in studies directed at elucidating how cells respond to cancer- relevant mechanical forces. The completed instrument design will be mature and ready for widespread adoption by cancer researchers, as well as by investigators from other fields of biomedical research.. Similar to how genetic perturbations or hits accumulate as tumors progress towards malignancy, physical changes in the tumor also accumulate and contribute directly to cancer cell growth, survival, and metastatic spread. The proposed instrument will provide new mechanistic insight into how cancer cells feel and respond to these physical stimuli. Basic prototypes of the instrument already have identified a nanoscale spatial perturbation that is linked to highly aggressive breast cancers. Maturation of the technology will facilitate the development of new therapeutics that target the physical basis of cancer.